Human epididymal expression sperm binding protein HEL-207 as well as encoding gene and application thereof

A technology of HEL-207, binding protein, applied in the direction of anti-animal/human immunoglobulin, application, gene therapy, etc., can solve the problem of poorly understood protein function, etc., to achieve the effect of less side effects

A technology of HEL-207, binding protein, applied in the direction of anti-animal/human immunoglobulin, application, gene therapy, etc., can solve the problem of poorly understood protein function, etc., to achieve the effect of less side effects

CN101724049AInactive Publication Date: 2010-06-09李建远
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  • Human epididymal expression sperm binding protein HEL-207 as well as encoding gene and application thereof
  • Human epididymal expression sperm binding protein HEL-207 as well as encoding gene and application thereof
  • Human epididymal expression sperm binding protein HEL-207 as well as encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1. Preparation of Human Epididymal Expression Protein HEL-207 by Genetic Engineering Technology

[0040] Gene cloning and sequence analysis

[0041] Large-scale sequencing and screening of the human epididymis cDNA library constructed in our laboratory was performed to obtain expressed sequence tags (EST), and electronic cloning was performed using the Unigene database to obtain the full-length cDNA of human HEL-207. Using Expasy Translate tool (http: / / us.expasy.orghttp: / / us.expasy.org), InterProScan (http: / / www.ebi.ac.uk / Tools / http: / / www.ebi.ac. uk / Tools / ) and Protein roteinblast (http: / / www.ncbi.nlm.nih.gov / BLAST / http: / / www.ncbi.nlm.nih.gov / BLAST / ) etc. to predict the peptide sequence of HEL-207 and assumed functions. SignalP (http: / / www.cbs.dtu.dkhttp: / / www.cbs.dtu.dk), PSORTII and WoLFPSORT (http: / / psort.nibb.ac.jp) and other software analyzes and predicts the signal peptide of the protein and intracellular localization. ProfileScan (http: / / myhits.isb-si...

Embodiment 2

[0045] Example 2. Preparation of anti-HEL-207 polyclonal antiserum

[0046] Polyclonal antibodies were prepared by immunizing BALB / C mice with recombinant HEL-207 protein. Briefly, each mouse was injected with 50 μg of recombinant protein and the same amount of complete Freund's adjuvant (CFA) on day 1. Then on the 15th, 30th and 45th days, 25 μg of recombinant protein and the same amount of incomplete Freund's adjuvant (IFA) were injected to boost the immunization. On the 60th day, blood was collected from the eyeball, and the serum was separated to analyze the titer and specificity of the antibody by ELASA and western blot. ELISA analysis showed that the antibody titer reached 1:10000. Western blot showed good specificity for both recombinant protein and native HEL-207 extracted from human epididymis fluid.

Embodiment 3

[0047] Example 3. Study on Human HEL-207mRNA Tissue Expression Profile

[0048] In order to determine the expression pattern of HEL-207, semi-quantitative RT-PCR was used to analyze the expression difference of HEL-207 gene in human epididymis head, body, tail, testis, heart, liver, spleen, lung, kidney, stomach and other tissues. Use TRIzol (Tiangen, Beijing, China) to extract total RNA, 1ug ​​total RNA uses 20UAMV reverse transcriptase (Promega) and 0.3ugoligod T 18 (Promega) was reverse transcribed into cDNA. Then, 2ul of the synthesized cDNA was amplified by PCR using gene-specific primers. 20ul reaction system contains: 2ul 10×PCR buffer (with MgCl 2 ), 2ul dNTP Mix (10mmol / L), 1ul each primer ul (25umol / L), 1ul Taq DNA polymerase (2.5U / ul), 2ulcDNA template and 11ulddH 2 O. The program of PCR reaction was: 94°C, 10min; 94°C, 1min; 54°C (for HEL-207) / 49°C (β-actin) for 30s; 72°C, 1min (35 cycles); 72°C extension for 7min. The expression of β-actin was used as an inte...

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Abstract

The invention belongs to the biotechnology and the medical field and discloses a new human epididymal specific expression sperm binding protein HEL-207 as well as preparation and application thereof. The invention discloses an amino acid sequence of the HEL-207 protein and provides a recombinant expression vector which carries a DNA sequence used for encoding the protein. The protein is located at the back part of the head of a mature sperm, an RT-PCR result indicates that the gene has expression in an epididymis tissue, a testis tissue, a cardiac tissue, a lung tissue, a liver tissue and a spleen tissue, and a band of about 46 KD is detected in an epididymis by using Western blot. The HEL-207 protein can be used as a target protein for developing an immunological contraceptive and clinically diagnosing and treating male infertility. A method for detecting a corresponding gene or protein based on development of the HEL-207 protein can be widely used for the research field of reproductive medicine.

Description

technical field [0001] The invention belongs to the field of biotechnology and medicine. Specifically, the present invention relates to a new human epididymis expressing sperm-binding protein HEL-207 and its coding gene and application Background technique [0002] The development of human beings in this century is facing two serious problems: on the one hand, the global population is rapidly expanding and is expected to reach 9 billion in 2050; on the other hand, according to the World Health Organization survey, about 15% of couples are infertile The problem is that the incidence of infertility in developed countries has soared in the past 10 years, and it can be as high as 30% in developed countries in Europe, of which male factors account for about half. The World Health Organization predicts that with the increase of environmental pollution and sexually transmitted diseases and other pathogenic factors, infertility will become the third major disease after tumors and c...

Claims

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Application Information

Patent Timeline
09 Jun 2010
Publication
CN101724049A
IPC
C07K14/435; C12N15/12; C12N15/63; C12N5/10; C07K16/18; A61K38/17; A61K31/7088; A61K48/00; A61K35/12; A61P15/16; A61P31/00; G01N33/53; A61K35/48
Inventors
李建远